Clinical Investigation
Radiation-Induced Lymphocyte Apoptosis to Predict Radiation Therapy Late Toxicity in Prostate Cancer Patients

https://doi.org/10.1016/j.ijrobp.2008.10.039Get rights and content

Purpose

To examine a potential correlation between the in vitro apoptotic response of lymphocytes to radiation and the risk of developing late gastrointestinal (GI)/genitourinary (GU) toxicity from radiotherapy for prostate cancer.

Methods and Materials

Prostate cancer patients formerly enrolled in a randomized study were tested for radiosensitivity by using a radiation-induced lymphocyte apoptosis assay. Apoptosis was measured using flow cytometry–based Annexin-FITC/7AAD and DiOC6/7AAD assays in subpopulations of lymphocytes (total lymphocytes, CD4+, CD8+ and CD4−/CD8−) after exposure to an in vitro dose of 0, 2, 4, or 8 Gy.

Results

Patients with late toxicity after radiotherapy showed lower lymphocyte apoptotic responses to 8 Gy than patients who had not developed late toxicity (p = 0.01). All patients with late toxicity had apoptosis levels that were at or below the group mean. The negative predictive value in both apoptosis assays ranged from 95% to 100%, with sensitivity values of 83% to 100%. Apoptosis at lower dose points and in lymphocyte subpopulations had a weaker correlation with the occurrence of late toxicity.

Conclusions

Lymphocyte apoptosis after 8 Gy of radiation has the potential to predict which patients will be spared late toxicity after radiation therapy. Further research should be performed to identify the specific subset of lymphocytes that correlates with late toxicity, followed by a corresponding prospective study.

Introduction

The goal of radiation therapy is to irradiate the tumor with a dose sufficient to kill all malignant cells while minimizing the damage to surrounding normal tissue. Dose is usually limited by the tolerance of the surrounding normal tissue (1). The effects of normal tissue toxicity can be detrimental. To minimize these effects, dose tolerance limits have been established from clinical data and set using population averages (2). Current radiotherapy guidelines typically limit dose such that the normal tissue late toxicity risk is less than 5% at 5 years posttreatment (TD5/5). Although these guidelines limit the number of patients with late effects, they do not eliminate these effects and moreover cannot predict which patients will experience toxicity. Furthermore, these limits do not take into account patients who have normal-tissue (and perhaps tumor-tissue) radiation resistance whereby average dose tolerance limitations may not be ideal for the greatest therapeutic gain. In this study, patients previously treated for carcinoma of the prostate were studied. Because of the anatomical location of the prostate, prostate cancer late toxicity affects both the gastrointestinal (GI) and genitourinary (GU) systems and can be particularly debilitating.

Even with rigid dose tolerance limits, patients respond with different levels of toxicity to a given treatment schedule (3). The development of a predictive assay would allow patients who are prone to late toxicity to consider the risk–benefit ratio of radiation therapy and perhaps be considered for an alternative type of treatment, such as surgery.

The causes of individual radiation sensitivity are still largely speculative. One hypothesis suggests that radiosensitive patients have an altered genetic makeup that causes cells to be less able to recognize or repair DNA damage 4, 5. In turn, these patients do not respond the same way to a course of radiation therapy. This could manifest as either an altered intrinsic apoptotic response in normal tissue or altered normal tissue function in the treatment volume as a consequence of repeated fractionated doses.

If radiation sensitivity is a genetic phenomenon, then these individuals may be influencing the averaged dose limits used in treatment regimens (6). Initial studies have suggested that if a predictive assay were developed to identify those patients sensitive to radiation, then the dose given to the remaining patients could be increased and perhaps lead to higher tumor control rates (7) without additional late toxicity.

Previous reports have suggested that peripheral blood lymphocyte apoptosis could be used to identify radiosensitive patients based on the apoptotic response of T lymphocytes to large in vitro doses. Ozsahin et al.8, 9 found that within a heterogeneous cancer patient population, a correlation existed between low levels of radiation-induced apoptosis in lymphocytes and late toxicity after radiation therapy. These investigators proposed that apoptosis in subpopulations of T lymphocytes (CD4+ and CD8+) could be used to identify radiation-sensitive patients before therapy.

The following study was conducted to test the postulate that reduced lymphocyte radiation sensitivity, as expressed by apoptosis, increases the risk of late toxicity. The specific aim was to demonstrate whether radiation-sensitive individuals could be identified within a homogeneous population of prostate cancer patients.

Section snippets

Patient population

Subjects were selected from a cohort of prostate cancer patients who participated in a prospective iridium implant study (10). This initial randomized study accrued 104 patients with T2 and T3, N0, M0 prostate cancer from 1992 to 1997. Patients were treated with either 66 Gy in 33 fractions delivered by external beam over 6.5 weeks or with an iridium implant of 35 Gy delivered over 48 hours followed by 40 Gy in 20 fractions over 4 weeks. Median follow-up at the time of publication was

Patient cohort

In total, 45 patients consented to participate in this study, of whom 7 (16%) had experienced late toxicity (GI or GU toxicity Grade ≥2). To test reproducibility, 7 randomly-selected patients had repeated blood samples collected. Elapsed time between these samples ranged from 2 to 12 months. The treatment type was equally represented, with 23 patients (51%) having iridium implants and EBRT and 22 (49%) with EBRT only. Of the 7 patients with late toxicity (radiation sensitive), two underwent

Discussion

The aim of this study was to identify patients at risk for late toxicity based on radiation-induced apoptotic response in lymphocytes. This work complements previous studies 8, 9 that used a similar, but not identical, assay in a heterogeneous patient population. The premise of the current study was that patients with increased late toxicity would display reduced levels of apoptosis when isolated lymphocytes were exposed to in vitro radiation. A proposed mechanism is that in patients

Conclusion

In conclusion, we have shown that there was significantly less lymphocyte apoptosis after exposure to 8 Gy in patients who had previously experienced late radiation toxicity than in those patients who had not. The possibility of using apoptosis as a predictive assay to identify patients who are at low risk for late toxicity is supported by this research. The negative predictive value of this approach appears high in specific subpopulations of lymphocytes (95–100%). Therefore, this tool may hold

Acknowledgments

The authors acknowledge the expert technical assistance of Nicole McFarlane for her outstanding skills in flow cytometry, Jan Barclay and the Clinical Trials department at the Juravinkski Cancer Centre for accruing and receiving consent from patients, and Ashley Hodgins for her technical assistance. The authors also acknowledge helpful discussion and insight from Dr. Nigel Crompton. This research was supported by the Canadian Association of Radiation Oncologists (Abbott–CARO Uro-Oncologic

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  • A Review of Radiation-Induced Lymphocyte Apoptosis as a Predictor of Late Toxicity After Breast Radiotherapy

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    Citation Excerpt :

    Two large prospective studies reported that CD8 lymphocytes had excellent NPV in detecting ≥ grade 3 late toxicities [22,29]. Sensitivity and specificity was 33% and 45% in CD4 cells and 67% and 55%, respectively, when tested in prostate patients [54]. Similarly, CD8 lymphocytes were also reported to have superior NPV and PPV compared with CD4 lymphocytes (91% and 19% vs. 81% and 9%, respectively) [22].

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Conflict of interest: none.

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